Gyros IncSupplier Diversity Partner REXXIP HN BUFFER 25 ML PER VI DFS Item

Protein isolation Bacteria - Borrelia burgdorferi

Experiment
Protein isolation Bacteria - Borrelia burgdorferi
Product
Gyros IncSupplier Diversity Partner REXXIP HN BUFFER 25 ML PER VI DFS Item from Fisher Scientific
Manufacturer
Fisher Scientific

Protocol tips

Upstream tips
- There is a previous cell lysate step.
Protocol tips
- Total cell lysate is subjected to ultracentrifugation (435,700 × g, 1 h, 4°C) to pellet membrane vesicles.

- Membrane pellets are rinsed in 1.0 ml of HN buffer with protease inhibitors, reisolated by ultracentrifugation, and suspended in 200 to 400 μl of HN buffer plus complete protease inhibitor cocktail with the aid of a coated homogenizer.
Downstream tips
- Protein precipitation is performed following Trichloroacetic Acid Solution, MP Biomedicals™ protocol (Fisher Scientific).

Publication protocol

Total cell lysate was subjected to ultracentrifugation (435,700 × g, 1 h, 4°C) in a Sorvall Discovery M150 SE Ultracentrifuge to pellet membrane vesicles. Membrane pellets were rinsed in 1.0 ml of HN buffer with the addition of protease inhibitors, reisolated by ultracentrifugation, and suspended in 200 to 400 μl of HN buffer plus complete protease inhibitor cocktail with the aid of a small Teflon coated homogenizer (Kontes, Vineland, NJ). Suspended membrane-associated proteins were stored at −80°C until use. Membrane protein quantities were determined by using a modified Lowry protein assay with bovine serum albumin as a standard.

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Discussion

Discussion

5 years ago

Author: Hollie Fowler United Kingdom

How can I deal with my pellet being too viscous?

I am using 8M Urea to lyse my cells but after centrifugation my pellet is very viscous. This causes me trouble when I try to collect the supernatant. Is there any way to avoid it and do you think it will have an effect on the concentration of my protein?

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Papers

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Manufacturer protocol

Download the product protocol from Fisher Scientific for Gyros IncSupplier Diversity Partner REXXIP HN BUFFER 25 ML PER VI DFS Item below.

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